Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive forms of human squamous cell carcinoma (SCC). ESCC patient prognosis remains poor due to late diagnosis and limited response to chemoradiotherapy, highlighting the need for novel therapeutic strategies in the treatmen of this disease. Tumor-initiating cells (TICs) are subsets of tumor cells possessing self-renewal and differentiation capabilities as well as enhanced chemoresistance, properties that have contributed to their emergence as cancer therapy targets. TICs, defined by high expression of CD44, the receptor for hyaluronic acid, have been identified in ESCC~ however, mechanisms regulating these subpopulations have yet to be elucidated. We have isolated CD44 High (CD44H) populations displaying characteristics of TICs from ESCC cells and demonstrated that transforming growth factor (TGF)-beta, promotes expansion of CD44H subpopulations concomitant with activation of Notch signaling. The Notch pathway is a context-dependent regulator of cell fate that is dysregulated in diverse cancer types~ however, the role of this pathway in ESCC is not yet known. In the current proposal, we hypothesize that Notch (N1) activation in the invasive front of ESCC acts in concert with TGF-beta to facilitate expansion of CD44H cell populations that are associated with the malignant properties of ESCC. We will test this through three interrelated specific aims. Aim 1 is to elucidate the role of N1 in expansion of CD44H ESCC cell populations. This will be achieved using inducible lentiviral overexpression and silencing systems to determine the requirement for N1 signaling in generation of CD44H cells in monolayer and 3-dimensional culture. These expression systems will also be used in concert with assays of migration, invasion, colony formation and xenograft tumor formation to assess the role of N1 in CD44H cell- mediated tumorigenicity. Aim 2 is to characterize the molecular mechanisms through which N1 and TGF- beta cooperate to promote CD44H cell expansion. Based on published findings and preliminary data, we hypothesize that TGF-beta- and N1-mediated signaling pathways may crosstalk to initiate a cell fate switch involving suppression of the N1 target gene Notch 3 (N3). We will test this by examining the influence of TGF-beta on N1-mediated transcriptional regulation of N3 using immunoprecipitation (IP), promoter activity assays and chromatin IP. We will also use inducible lentiviral-mediated overexpression and silencing systems to investigate the role o N3 signaling in generation of CD44H subpopulations. Aim 3 is to characterize the role of N1 in tumor growth and TIC expansion in vivo using genetically engineered mouse models. We will evaluate the role of N1 in ESCC progression by inactivating N1 in established ESCC lesions using inducible K5-ERT-Cre~Notch1floxf/lox mice and assessing effects on tumor progression and TIC content. Collectively, these studies will reveal mechanisms through which N1 supports ESCC progression while building a platform for new avenues toward translational applications for therapy of ESCC, which remains resistant to existing therapies.